Project Summary: Invasive fungal infections (IFI) constitute a rising threat to patients in the current practice of medicine. The modern physician uses potent immunomodulatory therapeutics to intervene on complex illnesses such as autoimmune disorders, malignancy and transplantation. While the impact of these therapeutics have resulted in major clinical breakthroughs, their immune inhibitory effects have profound side effects, often leaving patients at high risk for life-threatening infectious complications including IFI. To address this growing unmet need, we have turned attention to neutrophils, a critical first-responder innate immune cell required for rapid fungal elimination. In fact, patients with insufficient or dysfunctional neutrophils are at the highest risk for IFI. In macrophages, another innate immune cell, the recognition of the complex multi-layered carbohydrate fungal cell wall is performed, in part, through the lectin receptor, Dectin-1, which binds to ?-1,3- glucan and activates spleen tyrosine kinase (syk) resulting in upregulation of pro-inflammatory cytokine production. While syk activity is essential for fungicidal activity in macrophages, its role in neutrophils has yet to be defined. Several issues present major impediments towards defining the role of syk in neutrophils. First, neutrophils are short lived cells (<24hrs) and second, genetic manipulation is difficult owing to their terminal differentiation. Moreover, the generation of syk-deficient mice is not possible given syk contribution to embryonic vascular development. Despite these challenges, we sought to define the role of syk in neutrophil- fungal interactions through the following key observations. First, we address the longevity of neutrophils by using conditional activation of the transcription factor, HoxB8. Through expression of HoxB8, precursor stem cells or myeloblasts remain indefinitely dividing, and following HoxB8 inactivation, myeloblasts differentiate permitting generation of unlimited neutrophils. Second, we demonstrate that the HoxB8 system is amenable to CRISPR/Cas9 editing allowing generation of syk-deficient neutrophils. Third, in order to deconvolute the complex fungal surface, we developed fungal-like particles using purified fungal carbohydrates to probe specific neutrophil responses. Fourth, we demonstrate that HoxB8 neutrophils complement in vivo neutrophil function in a mouse model of neutropenia and Candida infection. To determine the molecular mechanism of syk in fungal innate immunity in neutrophils, we propose three independent specific aims: (1) Determine the in vitro response of syk-deficient HoxB8 neutrophils to defined microbial stimuli, (2) Define the role of syk in neutrophil effector function in response to yeast morphotype, and (3) Delineate the contribution of syk to neutrophil-fungal immunity in vivo. At the conclusion of our studies, we will clarify the subcellular mechanisms responsible for neutrophil-fungal pathogenesis with the hope to develop improved fungicidal strategies to address the growing clinical burden of invasive fungal disease, especially in the highest risk patients. !